Ionizing Radiation Induces Phosphorylation of Caveolin-1 on Tyr14

Abstract

Caveolin-1 (CAV1) is a 22 kDa membrane protein associated with caveolae in the cell membrane. It is involved in endocytosis, modulation of signal transduction, and has been reported to mediate radioprotection. Various stimuli such as oxidative stress, insulin, or osmotic shock are known to induce phosphorylation of caveolin-1 on Tyr14. This event is an important prerequisite for CAV1 function in the formation of caveolae and the interaction with multiple signaling molecules. The aim of the present study was to test if ionizing radiation (IR) induces phosphorylation of CAV1 on Tyr14 similar to oxidative stress, and to test the role of stress signaling kinases p38, Src family kinases (SFK), and c-Jun N-terminal kinase (JNK). The human lymphoblastoid cell line TK6, which does not express endogenous CAV1, was transduced with two different lentiviral vectors, containing either the cDNA of wtCAV1 or the cDNA of CAV1Y14F (substitution of Tyr by Phe on position 14) connected by the IRES element of EMCV with the cDNA of EGFP. Oxidative stress in TK6-CAV1 and TK6-CAV1Y14F was induced by treatment with 1 mM - 50 mM H2O2 for 20 min at 37 °C; cell irradiation was performed using 6 MV X-ray (6.7 Gy/min, Elekta Synergy). Cell lysates were prepared at different time points (5 min to 8 h) after IR. Specific kinase inhibitors were used to explore the functional relevance of kinase activation for CAV1 phosphorylation after H2O2 or IR. Phosphorylation was detected by Western blotting using phospho-specific antibodies, quantified and normalized to the unphosphorylated form. Mean values and SEM of at least three independent experiments were calculated. Phosphorylation of CAV1, p38, SFK and JNK in TK6-CAV1 was markedly increased by H2O2 concentrations in the range of 5 mM – 20 mM. Activation of p38, SFK and JNK was similar in TK6-CAV1Y14F and thus independent of Tyr14 phosphorylation of CAV1. Radiation doses of 2 Gy and 4 Gy clearly increased phosphorylation of CAV1, p38, SFK and JNK with different kinetics. While maximum CAV1 and SFK phosphorylation occurred early after irradiation (15 min – 1 h), activation of p38 and JNK peaked at the later time points (1 h – 3 h). Inhibition of p38, SFK and JNK revealed SFK as the major kinases for CAV1 phosphorylation on Tyr14 after induction of oxidative stress. Induction of CAV1 phosphorylation on Tyr14 and activation of stress-associated signaling molecules indicated the activation of similar signaling proteins in response to H2O2 treatment and IR with H2O2 -induced phosphorylation being more potent. Activation of SFK was an important factor for the phosphorylation of CAV1 after treatment with H2O2 in the TK6 cells used here. Studies on the role of the different kinases and phosphorylation of CAV1 regarding proliferation and apoptosis after irradiation are in progress.